The present study was conducted on corpus epididymis of twelve adult buffalo bulls, collected from slaughter house during different seasons of the year i.e. spring season, summer season, autumn season and winter season. The tissues were collected, fixed in 10% NBF and processed for paraffin sectioning. The sections of 5-6 μm were stained with different stains. The study revealed low epithelial height, small luminal and tubular diameter during summer season. The intercellular and intracellular vacoulations were abundantly present and more pronounced during summer season in the corpus epididymis than other seasons.
The buffalo bulls are capable of breeding throughout the year, but seasonal fluctuation in reproductive function is evident in most countries with semen quality being poorer in summer than in winter (Barile, 2005). The histomorphological studies have been conducted on epididymis in camel (Tingari and Moniem, 1979), goat (Goyal and Williams, 1991), buffalo (Singh and Roy, 1995), and sheep (Elzoghby et al., 2014) but very few reports were available on seasonal variation in structure of epididymis of buffalo (Arrighi et al., 2010; Ibrahim et al., 2013). Hence, the present study was planned to explore season changes in the epididymis of buffalo bulls.
Materials and Methods
The study was conducted on corpus epididymis of twelve adult buffalo bulls, divided into four groups, collected from slaughter house during different seasons of the year i.e. Group-I (Spring season, February-April), Group-II (Summer season, May-July), Group-III (Autumn season, August-October) and Group-IV (Winter season, November-January). The tissues were collected from body of epididymis and fixed in 10% NBF. After fixation, the tissues were processed for paraffin block preparation by acetone benzene schedule (Luna, 1968). The blocks were prepared and sections of 5-6 μm thickness were stained with hematoxylin and eosin, Masson’s trichrome, Verhoeff’s stain and Gridley’silver stain. The micrometrical observations were recorded.
Results and Discussion
The biometrical values of thickness of left and right corpus epididymis during different seasons have been summarized in Table 1. The thickness of corpus epididymis in left and right side during spring and summer season did not vary significantly (p > 0.05) from each other but varied significantly (p < 0.05) from autumn and winter season. Although the values of right epididymis within the same season were slightly higher than the left epididymis but the difference was non-significant (p > 0.05). The average thickness of corpus epididymis was significantly higher (p < 0.05) during autumn and winter as compared to spring and summer. Rind et al. (2006) reported that the thickness of body of left epididymis was 0.89 ± 0.22 cm while that of the right epididymis was 0.83 ± 0.21 cm.
Table 1: Mean ± SE thickness (mm) and breadth (cm) of left and right corpus epididymis during different seasons
|Spring||1.76 ± 0.17b||1.53 ± 0.10b||1.65 ± 1.10b||1.60 ± 0.07a||1.34 ± 0.11b||1.47 ± 0.07a|
|Summer||1.42 ± 0.14b||1.41 ± 0.29b||1.41 ± 0.15b||1.30 ±0.11b||1.20 ± 0.05b||1.25 ± 0.06b|
|Autumn||2.68 ± 0.26a||2.52± 0.22a||2.60 ± 0.16a||1.38 ± 0.04b||1.48 ± 0.08b||1.43 ± 0.04ab|
|Winter||2.72 ± 0.15a||2.59 ± 0.07a||2.65 ± 0.08a||1.56 ± 0.02a||1.69 ± 0.14a||1.62 ± 0.07a|
Mean value with atleast one same superscript, within column, didn’t differ significantly (p > 0.05)
The biometrical values of breadth of left and right corpus epididymis during different seasons have been summarized in Table 1. It was concluded that the breadth of left corpus during summer was significantly lower than in other seasons. The values of spring, summer and autumn season did not vary significantly (p > 0.05) from each other but breadth of right corpus region during winter season was significantly higher (p < 0.05) than other seasons. Although, in general the values of breadth of right epididymis within the same season were slightly higher than the left epididymis but the difference was non-significant (p > 0.05). The average breadth of the right corpus epididymis did not vary significantly (p > 0.05) during spring and winter. The average breadth of corpus epididymis was significantly lower (p < 0.05) during summer and significantly higher (p < 0.05) in winter season. The epididymal duct was surrounded by a thick layer of dense connective tissue, tunica albuginea covered by mesothelium. It was consisted of dense collagen fibres intermingled with few reticular fibres and very few smooth muscles as reported earlier by Goyal and Dhingra (1975), Singh (1989) and Gayake et al. (1997) in buffalo. Elastic fibres could not be observed in the tunica albuginea as reported by Singh and Dhingra (1971) however Singh (1989) reported elastic fibres in the epididymal capsule of buffalo. Large number of blood vessels and nerve fibres were observed close to tunica albuginea as reported by Singh (1989) in buffalo epididymis. Tunica submucosa was consisted of loose connective tissue in between the ducts/tubules and a dense tissue towards the periphery which blended with the tunica albuginea.
The corpus epididymis was lined by pseudostratified columnar having stereocilia on its apical surface. It consisted of mainly tall slender principal cells and polygonal basal cells, however other cells types observed in this study were apical cells, intraepithelial lymphocytes, macrophages and halo cells as reported by Abdou et al. (1985) and Singh and Roy (1995) in buffalo, Lopez et al. (1989) in stallion, Goyal and Williams (1991) and Sharma et al. (2014) in goat however no halo cells were reported by Sinowartz (1981), Goyal (1985) and Singh and Roy (1995) in their studies on bovine and buffalo epididymis. The principal cells with stereocilia were the main cell type in the epithelium having variable height and had oval or elongated nuclei which was basophilic in nature as reported by Goyal (1985) in bovine, Singh (1989) in buffalo and Mohamed (2005) in bovine epididymis. During summer season, the nuclei were not dense and gave a vacuolated appearance. The nuclei of the basal cells were located in close contact with the basement membrane of the epithelium. Basal cells were mostly pyramidal to hemispherical in shape as reported earlier by Mohamed (2005) in bovine epididymis. Halo cells were scattered throughout the epididymal epithelium. These were small cells with a heterochromatic round nuclei and scanty lightly stained cytoplasm. These cells have been named as intraepithelial lymphocytes (Sinowatz, 1981; Goyal 1985; Mohamed 2005). Halo cells had a distinct ring around them and were observed near the basal cells just above the basement membrane. They were round in shape. Halo cells were more abundant during winter season.
Intercellular and intracellular vacuolations were abundantly present in corpus epididymis (Fig. 1). These vacoulations were more pronounced during summer season than other seasons. Some dead and degenerated spermatozoa could be observed in these vacoulations. This feature indicated the lysosomal activity of the principal cells. Epithelial height of corpus epididymis was 53.66 ± 1.60 µm, 50.74 ± 1.47 µm, 55.58 ± 1.79 µm and 59.33 ± 1.38 µm during spring, summer, autumn and winter seasons respectively. It was observed that epithelium height in winter season was significantly higher (p < 0.05) than in spring and summer and seasons. Arrighi et al. (2010) also observed that epithelial cell height reduced during the non-mating summer months of the corpus epididymis in buffalo and Ibrahim and Singh (2014) reported in buffalo that mean corpus epithelial height was 51.71 ± 4.49 µm in winter and 48.92 ± 2.04 µm in summer. Lamina proria was a thin layer of loose connective tissue consisting of collagen fibres, fine reticular fibres, fibroblasts and fibrocytes (Fig. 2).
|Fig. 1: Photomicrograph of corpus epididymis during summer showing numerous vacuoles (arrow). Hematoxylin and Eosin x 100||Fig. 2: Photomicrograph of corpus epididymis during summer showing collagen fibres. Masson’ Trichrome x 100|
Elastic fibres could not be demonstrated in this layer. Large number of subepithelial capillaries could be observed as reported earlier by Mohamed (2005) in bovine epididymis and these capillaries were more numerous during winter season. The capillaries were invaginating into epithelial cells but Sinowatz (1981) has observed electron microscopically that these capillaries remained separated from epithelial cells by a distinct basement membrane and never projected in the epithelial cells. During winter and autumn seasons this layer was very compact and could hardly be observed. Smooth muscle cells were circularly arranged around the epididymal tubules. Micrometrical observations on thickness of the smooth muscle layer around tubules in corpus epididymis has been summarized in Table 2.
Table 2: Mean ± SE (µm) Micrometrical observations on corpus epididymis
|Parameters||Tubular diameter||Luminal diameter||Epithelium||Peritubular Muscle Layer|
|Spring||340.50 ± 15.39ba||253.85±11.36a||53.66 ± 1.60b||23.31 ± 0.93ba|
|Summer||321.18 ± 7.27b||244.62 ± 7.038a||50.74 ± 1.47b||25.31 ± 0.72a|
|Autumn||363.51 ± 13.33a||260.60 ± 9.70a||55.58 ± 1.79ba||23.44 ± 0.64ba|
|Winter||364.90 ± 18.49a||270.49 ± 7.70a||59.33 ± 1.38a||22.28 ± 1.04b|
Mean value with at least one same superscript, within column, do not differ significantly (p > 0.05)
The values during summer season were significantly higher (p < 0.05) than winter season but non-significant (p > 0.05) difference was observed in other seasons. Mohamed (2005) observed that in bovine epididymis the morphometric value of peritubular muscle layer from segment I to VI was 26.01 ± 1.76 µm, 24.99 ± 2.64 µm, 25.80 ± 2.42 µm, 27.80 ± 1.66 µm, 30.99 ± 1.42 µm and 55.60 ± 9.05 µm respectively and suggested the role of peritubular smooth muscle layer in rhythmic contraction which help in the propulsion of spermatozoa out of the lumen into the ductus deferens. Ibrahim and Singh (2014) also reported that the circumtubular muscle coat increased in thickness during summer season (26.10 ± 4.50 µm) and decreased during winter (19.62 ± 4.20 µm).
|Fig. 3: Photomicrograph of Corpus of epididymis in winter season showing Halo cells (arrow). Hematoxylin and Eosin X 10|
The lumina of the corpus epididymis in general was oval to triangular in shape as earlier observed by Mohamed (2005) in bovine epididymis. The spermatozoa content was more or less arranged in a circular fashion which depicts the circular motility of the mature spermatozoa stored in the cauda epididymis as earlier reported by Singh (1989) in buffalo bull. Diameter of lumen in corpus region was 253.85 ± 11.36 µm, 244.62 ± 7.038 µm, 260.60 ± 9.70 µm and 270.49 ± 7.70 µm during spring, summer, autumn and winter seasons respectively. Although the values were less during summer and spring seasons but the difference was non-significant (p > 0.05). However, Ibrahim and Singh (2014) observed corpus luminal diameter as 167.94 ± 18.09 in winter and 195.38 ± 18.43 µm during summer. The diameter of tubules during summer was significantly low (p < 0.05) as compared to autumn and winter seasons. Arrighi et al. (2010) also observed that the tubular diameter of the corpus epididymis was significantly reduced in summer.
It may be concluded that during summer rising temperature may have an impact on histomorphology of epididymis which can be a reason of low reproductive performance of buffalo bull in summer as compared to other seasons.